JBIR-137 and JBIR-138, new secondary metabolites from Aspergillus sp. fA75

Natural products are considered to be good sources for the screening of lead compounds of clinical drugs. We performed many drug screenings employing a variety of assay systems with crude extracts of microbial cultures as a traditional natural product library. In some assay systems, effective application of our crude extract library was difficult without making some improvements. From this viewpoint, we started to construct a purified natural compounds library from cultures of microorganisms. To achieve this, we established the high-throughput detection system for microbial secondary metabolites using UPLC-UV-evaporative light-scattering (ELS)-MS system, and more than 1000 compounds, which we have already isolated, were analyzed by the common analytic method and in our database. The registered compounds in microbial cultures are automatically identified with our system, which allows us easily to pick up unregistered compounds. The unregistered compounds are isolated from the cultures and store in our library. Because Aspergillus species are known to produce more than 950 documented bioactive compounds such as mevinolin, aflatoxin and citrinin,¹ their secondary metabolites are an important source to obtain various bioactive compounds. Therefore, we attempted to obtain secondary metabolites from cultures of Aspergillus. During chemical screening based on our analytic system, we isolated a new janthitrem derivative named JBIR-137 (1) and a novel metabolite JBIR-138 (2), together with the known compounds, a tremorgenic agent janthitrem B² and 6-hydroxycyclopiamine B³ from the culture of Aspergillus sp. fA75 (Figure 1). This paper describes the fermentation, isolation, structural elucidation, and briefly, biological activity of 1 and 2.

Figure 1

Structures of 1, 2, janthitrem B and 6-hydroxycyclopiamine B.

Aspergillus sp. fA75 was isolated from a soil sample collected in the forest at Noda, Chiba Prefecture, Japan. The strain was cultivated in 50-ml test tubes, each containing 15 ml potato dextrose medium (24 g l^–1; BD Biosciences, San Jose, CA, USA). The test tubes were shaken reciprocally (320 r.p.m.) at 27 °C for 2 days. Aliquots (4 ml) of the culture were transferred to 500-ml Erlenmeyer flasks containing brown rice 15 g (Akitakomachi, Yamagata, Japan), bacto–yeast extract 30 mg (BD Biosciences), sodium tartarate 15 mg, K₂HPO₄ 15 mg and water 45 ml. The flasks were incubated statically at 27 °C for 14 days.

The culture (10 flasks) was extracted with 80% aq. Me₂CO (200 ml per flask), and the extract was filtered. After concentration in vacuo, the aqueous residue was extracted with EtOAc (600 ml × 3). The EtOAc layer was dried over anhydrous Na₂SO₄ and evaporated in vacuo, yielding a dark brown gum (815 mg). The extract was fractionated using normal-phase medium-pressure liquid chromatography (Purif-Pack SI-30, Shoko Scientific Co., Yokohama, Japan) with a gradient system of n-hexane–EtOAc (0–25% EtOAc) followed by the stepwise solvent system of CHCl₃–MeOH (0, 2, 5, 10, 20, 30 and 100% MeOH) to obtain five fractions (5% fraction-1, 5% fraction-2, 5% fraction-3, 10 and 20–30%). The fractions were monitored by UPLC-UV-ELS-MS system. Compound 1 was isolated from the 5% MeOH fraction-1 (26.5 mg) by reversed-phase HPLC using a CAPCELL PAK C18 MG II column (5.0 μm, 20 i.d. × 150 mm; Shiseido, Tokyo, Japan) with 85% aq. MeOH containing 0.1% formic acid (flow rate 10 ml min^–1, Retention time (Rt)=11.6 min). From the 5% MeOH fraction-2 (89.1 mg), janthitrem B² (5.0 mg) was isolated by HPLC preparation. The 5% MeOH fraction-3 (45.6 mg) was applied to gel filtration chromatography (Sephadex LH-20, GE Healthcare BioSciences AB, Uppsala, Sweden) eluting with CHCl₃–MeOH (1:1) to yield crude 2 (45.6 mg). The obtained material was further purified by the HPLC (40% aq. MeOH containing 0.1% formic acid, Rt=17.3 min) to yield pure 2 (9.9 mg). The 6-hydroxycyclopiamine B³ (3.7 mg) was purified from the 20–30% MeOH fraction (124.7 mg) using LH-20 column chromatography and HPLC.

JBIR-137 (1) was obtained as a colorless amorphous solid: [α]²²-66 (MeOH; c 0.13); UV λ_max nm (ɛ): 281 (30 300), 290 (32 200) and 374 (5800) in MeOH; IR (ν_max): 3400 (hydroxy), 1618, 1455, 1371 (aromatic and pyrrole) cm⁻¹. Its molecular formula was determined to be C₃₇H₄₅NO₄, with 16 index of hydrogen deficiency by high-resolution ESI-MS (m/z 566.3273 [M–H]⁻, calcd for C₃₇H₄₄NO₄: 566.3270). The ¹H-, ¹³C- and heteronuclear single-quantum coherence NMR data (Table 1) showed 37 carbon signals including 7 methyls, 6 methylenes (one olefinic), 9 methines (3 oxygen-bearing, 2 aromatic and 3 olefinic) and 15 quaternary carbons (5 sp³ and 10 sp²). The planar structure of 1 was clarified on the basis of double-quantum-filtered COSY and constant time-HMBC (CT-HMBC)⁴ experiments (Figure 2a), as described below.

Table 1 ¹³C and ¹H NMR spectroscopic data for JBIR-137 (1) and JBIR-138 (2)

Figure 2

(a) Key COSY and HMBC correlations of 1. COSY: bold line; HMBC (¹H to ¹³C): solid arrow (J_CH=8 Hz) and dashed arrow (J_CH=3 Hz). (b) Partial relative configuration of 1. (NOESY correlation: arrow) (c) Key COSY and HMBC correlations of 2. COSY: bold line; HMBC: solid arrow (strong) and dashed arrow (weak). (d) Key NOESY correlations of 2.

In the CT-HMBC spectrum, ¹H–¹³C long-range correlations from a singlet methyl proton H₃-36 (δ_H 1.78) to the oxymethine carbon C-9 (δ_C 80.3), olefinic quaternary carbon C-34 (δ_C 143.0) and exo-methylene carbon C-35 (δ_C 111.5) proved the presence of an isopropenyl group. The sequence from H-9 (δ_H 3.86) to the olefinic methine H-11 (δ_H 5.71, δ_C 118.3) through the oxymethine proton H-10 (δ_H 3.96, δ_C 64.0) was observed in the COSY spectrum. In addition to the ¹H spin system, HMBC correlations from H-9 to the oxymethine carbon C-7 (δ_C 74.8) and C-11, from H-10 to the olefinic quaternary carbon C-12 (δ_C 149.4), and from H-11 to C-7 and C-12 revealed a six-membered ether ring (ring H). In consideration of all the above correlations, the structure of ring H was determined to be a 3,6-dihydro-2H-pyran bearing an oxygen and an isopropenyl group at C-10 and C-9, respectively. The ¹H–¹H spin systems from methylene protons H₂-5 (δ_H 2.61, δ_H 1.65) to the oxymethine proton H-7 (δ_H 4.62) through methylene protons H₂-6 (δ_H 2.18, δ_H 1.93), together with the HMBC correlations from H-11 to the oxygenated quaternary carbon C-13 (δ_H 77.6), from H₂-6 and H₂-5 to the quaternary carbon C-4 (δ_C 43.5), and from a singlet methyl proton H₃-33 (δ_H 1.03) to C-4, C-5 (δ_C 27.9) and C-13, indicated that ring G is a cyclohexane bearing an oxygen and a methyl group at C-13 and C-4, respectively. A ¹H–¹H spin-coupling system from methylene protons H₂-14 (δ_H 1.95, δ_H 1.66) to methylene protons H₂-17 (δ_H 2.64, δ_H 2.36) through methylene protons H₂-15 (δ_H 2.05, δ_H 1.66) and a methine proton H-16 (δ_H 2.80) was observed. ¹H–¹³C long-range couplings from a singlet methyl proton H₃-32 (δ_H 1.30) to the aromatic quaternary carbon C-2 (δ_C 154.5), quaternary carbons C-3 (δ_C 51.8), C-4 and the methine carbon C-16 (δ_C 50.7), from the methylene protons H₂-14 to the oxymethine carbon C-13, and from the methylene protons H₂-17 to the aromatic quaternary carbons C-2 and C-18 (δ_C 117.6), indicated the ring moieties E and F.

Strong m-couplings from the aromatic proton H-20 (δ_H 7.11) to aromatic carbons C-29 (δ_C 128.6) and C-31 (δ_C 139.2), and from the aromatic proton H-30 (δ_H 7.48) to aromatic carbons C-19 (δ_C 126.0) and C-21 (δ_C 136.9), allowed the assignment of a benzene-ring substructure (ring C). The ¹H–¹³C long-range couplings from the aromatic proton H-20 to C-18 and C-19 indicated that the benzene-ring moiety was substituted at C-18.

The remaining substructures were established as follows. Singlet methyl protons H₃-37/H₃-38 (δ_H 1.51) were long-range coupled to each other and to the oxygenated quaternary carbon C-24 (δ_C 74.6) and aromatic quaternary carbon C-23 (δ_C 141.6). Another set of singlet methyl protons H₃-39/H₃-40 (δ_H 1.44) were long-range coupled to each other and to the oxygenated quaternary carbon C-26 (δ_C 74.0) and the aromatic methine carbon C-27 (δ_C 128.2). The aromatic methine proton H-27 (δ_H 6.47) was long-range coupled to the aromatic carbons C-23, C-28 (δ_C 135.2) and C-29, revealing the sequence from C-24 to C-26 through C-23, C-28 and C-27, and showed the substituted position of C-28 to be at C-29. In addition to these correlations, ¹H–¹³C long-range couplings from the aromatic proton H-22 (δ_H 6.36) to aromatic carbons C-21, C-23, C-28 and C-29 indicated the formation of a 5-membered ring structure (ring B). The HMBC correlations from the aromatic protons H-20 and H-30 to the aromatic carbons C-22 and C-28, respectively, also supported these connections. Finally, the molecular formula of 1 and the ¹³C chemical shifts at C-24 and C-26 indicated a 2,2,6,6-tetramethyl-3,6-dihydro-2H-pyran and an indole-like moieties (rings C and D). Thus, the planar structure of 1 was revealed as shown in Figure 1. The structure of 1 is closely related to the janthitrems that were reported as tremorgenic mycotoxins.⁵

The partial relative configuration was determined from the NOESY spectrum and the corresponding coupling constants. A small coupling constant between H-9 and H-10 (<1 Hz) and strong NOE between H-9 and H-10 determined the relative configuration of the ring H as shown in Figure 2b. In the same manner, the NOESY correlations among H-9, H-10, H-7, H-5a, H-32 and H-15a indicated that these protons are located on the same side in the molecule. On the other hand, the NOEs among H-33, H-6b, H-14b and H-16 showed that these protons are on the opposite side from CH₃-32. Consequently, the relative configuration of ring E-H was established as shown in Figure 2b.

JBIR-138 (2) was obtained as a colorless amorphous solid: [α]²²_D –31 (MeOH; c 0.5); UV λ_max nm (ɛ): 230 (13 900) in MeOH. The molecular formula of 2 was established as C₁₉H₂₄O₇ (index of hydrogen deficiency=8) by high-resolution ESI-MS (m/z 363.1447 [M–H]⁻, calcd for C₁₉H₂₃O₇ 363.1444). The IR absorption (ν_max 3446, 1766, 1724 and 1674 cm⁻¹) indicated the presence of hydroxy, γ-butyrolactone, carboxylic acid and α,β-unsaturated ketone functional groups. The assignments of the ¹H and ¹³C NMR spectroscopic data were tabulated in Table 1.

The COSY spectrum showed a sequence from methylene protons H₂-1 (δ_H 2.34, δ_H 1.73) to methylene protons H₂-3 (δ_H 1.96, δ_H 1.05) through methylene protons H₂-2 (δ_H 1.60, δ_H 1.55), and a ¹H spin coupling between a methine proton H-9 (δ_H 3.14) and a methylene proton H-11a (δ_H 4.63). The CT-HMBC spectrum showed ¹H–¹³C long-range correlations (Figure 2c) as follows: from a singlet methyl proton H₃-14 (δ_H 1.23) to an sp³ quaternary carbon C-4 (δ_C 37.2), a methylene carbon C-3 (δ_C 36.1), a methine carbon C-5 (δ_C 55.5) and an oxymethylene carbon C-15 (δ_C 66.6); from H₂-2 to C-4 and a quaternary carbon C-10 (δ_C 49.0); and from H₂-1 to C-10 and C-5. These correlations indicated the presence of a cyclohexane ring bearing a methyl group and an oxymethylene group at the C-4 position. The strong HMBC correlations from the allylic methyl proton H₃-12 (δ_H 1.99) to the olefinic methine carbon C-7 (δ_C 131.1), the deshielded olefinic quaternary carbon C-8 (δ_C 156.7), and the methine carbon C-9 (δ_C 54.1), and weak correlations from H₃-12 and the olefinic methine proton H-7 (δ_H 5.91) to the α,β-unsaturated ketone carbonyl carbon C-6 (δ_C 197.4), showed a sequence from C-6 to C-9. The direct connectivity between C-5 and C-6 was revealed by HMBC correlations from the singlet methine proton H-5 (δ_H 2.82) to C-6 and C-7, which showed an octalone moiety. The ¹H–¹H spin coupling between H-9 and H-11a together with the common ¹H–¹³C long-range couplings from these protons to C-8, C-10 and the carbonyl carbon C-13 (δ_C 178.9) indicated a γ-lactone moiety, the presence of which was supported by the IR absorption at 1766 cm⁻¹ vide ante. Additionally, the long-range correlation from H-1 to C-13 demonstrated the direct connectivity between C-13 and C-10, establishing the condensation of the octalone and γ-butyrolactone ring moieties. Thus, a sesquiterpene structure was determined (Figure 2c). A succinic acid moiety was elucidated from the HMBC correlations from methylene protons H₂-2′ (δ_H 2.60) and H₂-3′ (δ_H 2.62) to carbonyl carbons C-1′ (δ_C 174.3) and C-4′ (δ_C 176.2). An ester linkage between C-15 and C-1′ was revealed by the HMBC correlation from the oxymethylene protons H₂-15 (δ_H 5.15, δ_H 4.66) to C-1′. Thus, the gross structure of 2 was elucidated (Figure 1).

The relative configuration of the sesquiterpene moiety of 2 was determined by the NOESY spectrum. NOESY correlations among H-1b, H-3b, H-5, H-9 and H-14 suggested that these protons are on the same face of the molecule. The correlations between H-1a and H-11a implied the relative configuration of the γ-butyrolactone ring shown in Figure 2d. To the best of our knowledge, the backbone of 2 has not yet been reported as a secondary metabolite produced by microorganisms.

The cytotoxic activities of 1, 2, janthitrem B and 6-hydroxycyclopiamine B against human ovarian adenocarcinoma SKOV-3 cells were examined by using the WST-8 ((2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) colorimetric assay (Cell Counting Kit; Dojindo, Kumamoto, Japan). After administered the compounds for 72 h, 1 and janthitrem B exhibited weak cytotoxic activities against SKOV-3 cells with the IC₅₀ of 12.5 and 37.6 μM, respectively. To the contrary, 2 and the 6-hydroxycyclopiamine B did not show cytotoxicity (IC₅₀>100 μM). Although janthitrem B and its derivatives were known as tremorgenic agents, their cytotoxic effects have not been reported. Further studies on biological activities of 1 are under way.